Glucokinase, a key regulatory enzyme in the hepatic and beta-cell glycolytic pathway, is a member of the hexokinase family of enzymes. However, almost nothing is known about the structure/function relationships of this enzyme family. Recently a number of missense mutations in glucokinase have been identified which are linked to early- onset non-insulin dependent diabetes mellitis. The overall goal of this proposal is to increase our understanding of the molecular basis for catalysis and regulation of human liver and beta-cell glucokinases and to define the molecular mechanism by which mutations in glucokinase lead to glucose intolerance in noninsulin dependent diabetes mellitus. It is proposed to overexpress the human liver and beta-cell glucokinases in a bacterial expression system and to characterize their kinetic and physical properties. It is also proposed to attempt to obtain the x-ray crystal structure of the human liver and beta-cell isoforms and of mutants of these enzymes. Classical chemical modification will be used to study structure/function relationships. It is also proposed to use site-directed mutagenesis to identify those residues involved in catalysis and substrate binding and to determine the consequences of mutations in glucokinase found in MODY and other NIDDM patients. In order to determine the consequences of mutations in human glucokinases on hepatic glucose metabolism, wild-type and mutant forms of glucokinase will be expressed in hepatoma cells and the amount and type of enzyme will be related to rates of glycolysis/gluconeogenesis and glycogen synthesis. The hypothesis that glucokinase mutations function as dominant regulators of transporter activity will also be tested by overexpressing wild-type and mutant glucokinases in AtT-20ins cells. Mutated forms of glucokinase will also be expressed in transgenic mice to determine whether these mutations per se affect insulin secretion and/or hepatic metabolism in vivo. The structural basis for glucose-6- phosphate inhibition of low Km hexokinases will be investigated by kinetic analysis of chimeric constructs of hexokinase and glucokinase and of the N-terminal and C-terminal halves of low Km hexokinases. Elucidation of the structure/function relationships of human glucokinase and analysis of mutations found in human patients will enhance our understanding of the genetic basis of NIDDM and point the way to other genetic defects, as well as provide new insights into basic mechanisms for regulation of carbohydrate metabolism and ultimately, perhaps, new modalities for treatment of NIDDM.